bioRxiv preprint doi: https://doi.org/10.1101/482422; this version posted December 1, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license. APOE4 is Associated with Differential Regional Vulnerability to Bioenergetic Deficits in Aged APOE Mice Tal Nuriel1,2,*, Delfina Larrea1,3, David N. Guilfoyle4, Leila Pirhaji5, Kathleen Shannon6, Hirra Arain1,2, Archana Ashok1,2, Marta Pera1,3, Qiuying Chen7, Allissa A. Dillman8, Helen Y. Figueroa1,2, Mark R. Cookson8, Steven S. Gross7, Ernest Fraenkel5,9, Karen E. Duff1,2,10, †, and Estela Area-Gomez1,3, † 1Taub Institute for Research on Alzheimer's Disease and the Aging Brain, Columbia University, 630 West 168th Street, New York, NY 10032, USA. 2Department of Pathology and Cell Biology, Columbia University, 630 West 168th Street, New York, NY 10032, USA. 3Department of Neurology, Columbia University, 630 West 168th Street, New York, NY 10032, USA. 4Center for Biomedical Imaging and Neuromodulation, Nathan S. Kline Institute, Orangeburg, NY 10962, USA. 5Department of Biological Engineering, Massachusetts Institute of Technology, 350 Brookline Street, Cambridge, MA 02139, USA. 6Animal Facility, Nathan S. Kline Institute, Orangeburg, NY 10962, USA. 7Department of Pharmacology, Weill Cornell Medical College, 1300 York Avenue, New York, NY 10065, USA. 8Cell Biology and Gene Expression Section, Laboratory of Neurogenetics, National Institute on Aging, National Institutes of Health, Bethesda, Maryland, 20892 9Broad Institute, 415 Main Street, Cambridge, MA 02142, Cambridge, Massachusetts, 02139, USA. 10Division of Integrative Neuroscience in the Department of Psychiatry, New York State Psychiatric Institute, 630 West 168th Street, New York, NY 10032, USA. * Corresponding author: Tal Nuriel, email: [email protected] †These two authors contributed equally to this work and are co-senior authors. bioRxiv preprint doi: https://doi.org/10.1101/482422; this version posted December 1, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license. ABSTRACT The ε4 allele of apolipoprotein E (APOE) is the dominant genetic risk factor for late-onset Alzheimer’s disease (AD). However, the reason for the association between APOE4 and AD remains unclear. While much of the research has focused on the ability of the apoE4 protein to increase the aggregation and decrease the clearance of A, there is also an abundance of data showing that APOE4 negatively impacts many additional processes in the brain, including bioenergetics. In order to gain a more comprehensive understanding of the APOE4’s role in AD pathogenesis, we performed a multi-omic analysis of APOE4 vs. APOE3 expression in the entorhinal cortex (EC) and primary visual cortex (PVC) of aged APOE mice. These studies revealed region-specific alterations in several bioenergetic pathways, including oxidative phosphorylation (OxPhos), the TCA-cycle and fatty acid metabolism. Follow-up analysis utilizing the Seahorse platform revealed decreased mitochondrial respiration in the hippocampus and cortex of aged APOE4 vs. APOE3 mice, but not in the EC of these mice. Additional studies, as well as the original multi- omic data suggest that bioenergetic pathways in the EC of aged APOE mice may be differentially regulated by APOE4 expression. Given the importance of the EC as one of the first regions to be affected by AD pathology in humans, this differential bioenergetic regulation observed in the EC vs. other brain regions of aged APOE4 mice may play an important role in the pathogenesis of AD, particularly among APOE4 carriers. INTRODUCTION Possession of the ε4 allele of apolipoprotein E (APOE) is the major genetic risk factor for late-onset Alzheimer’s disease (AD). In normal physiology, the apoE protein plays a vital role in the transport of cholesterol and other lipids through the bloodstream, as well as within the brain (1-3). Although the three common isoforms of apoE (E2, E3 and E4) differ from each other by only two amino acids—apoE2 (Cys112, Cys158), apoE3 (Cys112, Arg158), apoE4 (Arg112, Arg158)—this small change in amino acid bioRxiv preprint doi: https://doi.org/10.1101/482422; this version posted December 1, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license. sequence has a large effect on protein structure, causing the apoE4 protein to possess a more globular structure due to increased interaction between its N- and C-terminal domains. This difference in structure, in turn, affects the type of lipids that each isoform binds, as well as the affinity of each isoform for the numerous receptors that mediate the uptake of apoE and its cargo into cells (see reviews by (1, 4)). Importantly, these isoform differences also have a major impact on the pathogenesis of late-onset AD. Although the APOE2, APOE3 and APOE4 alleles are present at a relative frequency of about 8%, 77% and 15% in the normal population (5, 6), the APOE4 allele is present at a relative frequency of 36-64% in AD patients (7-10), with individuals who possess one or two APOE4 alleles having a 3- or 12-fold increased risk of developing AD, respectively (11). Although a number of mechanisms have been proposed to help explain this APOE4-associated susceptibility to AD, the precise cause remains a source of debate. The prominent hypothesis is that this increase in AD risk is due to the ability of apoE4 to increase the aggregation and decrease the clearance of A (12-18). However, APOE4 expression has also been shown to have deleterious effects on numerous A-independent pathways, including lipid metabolism, tau pathology, bioenergetics, neuronal development, synaptic plasticity, the neuro-vasculature, and neuro- inflammation [see reviews by (19) and (20)], any number of which could play a pivotal role in the pathogenesis of AD among APOE4 carriers. In terms of APOE4’s effects on bioenergetics, a number of pivotal reports have demonstrated that APOE4 expression leads to widespread dysregulation of the brain’s bioenergetic capacity. For example, early reports by Eric Reiman and colleagues demonstrated that both young and old APOE4 carriers display decreased glucose utilization, as measured by fluorodeoxyglucose positron emission tomography (FDG- PET), in similar brain regions to that seen with AD patients (21-23). Additional reports detail the wide- range of bioenergetic insults that APOE4 expression can cause in the brain, including impaired insulin signaling (24, 25), reduced cerebral blood volume and cognitive function in response to a high fat diet (HFD) (26, 27), altered genetic expression of glucose-regulating enzymes and transporters (28, 29), and the generation of a toxic C-terminal fragment of apoE4 that can directly target electron transport chain (ETC) bioRxiv preprint doi: https://doi.org/10.1101/482422; this version posted December 1, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license. complexes in the mitochondria (30-32). In order to study the diverse effects that APOE4 expression has in the brain, we performed an initial multi-omic study of differential APOE isoform expression in an AD-vulnerable vs. an AD-resistant brain region from aged APOE targeted replacement mice, which express human APOE in place of their mouse Apoe gene and do not develop overt AD pathology. The EC was chosen as our AD-vulnerable brain region because it is one of the first brain regions to develop AD-related tau pathology, in humans (33, 34), and also because the morphology of the EC has been shown to be specifically affected by APOE4 gene expression. (35, 36). The PVC was chosen as our AD-resistant region because it is relatively spared in AD (33, 37, 38). As described below, these studies revealed APOE4-specific alterations in the levels of numerous genes and small-molecules related to energy metabolism, which were explored further using the Seahorse platform. In agreement with previous data, this analysis revealed APOE4-associated deficits in mitochondrial respiration in several brain regions. However, contrary to what we observed in other brain regions, the EC displayed increases in oxidative metabolism in the context of APOE4 expression, suggesting that, under metabolic stress, brain region-specific counterbalancing mechanisms are in play in the EC of aged APOE4 mice. Additional studies and further mining of the multi-omic data was then used to elucidate this differential bioenergetic regulation in the EC of aged APOE4 mice, which may play a causative role in the pathogenesis of AD and may point to novel therapeutic interventions for preventing or slowing the disease. RESULTS Multi-omic analysis reveals differential expression of energy-related genes and metabolites in the EC of aged APOE4 mice In order to investigate the effects of APOE4 expression in an untargeted manner, we performed a multi- omic analysis (transcriptomics and metabolomics) on RNA and small-molecules extracted from an AD- vulnerable brain region (the EC) vs. an AD-resistant brain region (the PVC) of 14-15 month-old APOE bioRxiv preprint doi: https://doi.org/10.1101/482422; this version posted December 1, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license.